The invention concerns chemically modified and activated hydroxyl-group-containing natural and synthetic, polymer solid body surfaces, which are employed for the binding of proteins, nucleic acids, low-molecular ligands, cells, microorganisms and other biological materials in fields of biology, biotechnology, as well as medicine, for analytical and preparative purposes, as well as a process for the production thereof.
Carrier-bound biologically active substances have been employed on a broad scale for many years, for the separation and isolation of specific recriprocally-acting partners. See: H. D. Orth, W. Brumer: Angew, Chem. 84 (1972) 319; W. B. Jakob, M. Wilchek (hrsg.): Methods in Enymol. 34 (1974), for binding of enzymes and use as ensyme reactors (K. Mosbach Ohrsg.) Methods in Enzymol. 44 (1976), as well as for qualitative and quantitative display detection of biologically significant compounds in biology, biotechnology, and also increasingly in medicine. In general, the following requirements are placed upon the so-called ideal matrix:
insolubility PA1 macroporosity PA1 mechanical and chemical stability PA1 particular form PA1 hydrophility PA1 low non-specific binding PA1 resistance to microbial and enzymatic influences PA1 presence of functional groups for chemical modifiction and activation
Such an ideal matrix, satisfying all of these requirements and thereby universally employable, does not exist. Therefore, in the last several years, a plurality of carrier materials has been described and is commercially available, including natural polymers (polysaccarides), such as dextranes and agarose, also synthetic polymers (such as polyvinyl alcohols, acrylic acid derivatives, vinyl polymers, as well as copolymers of natural and synthetic polymers, such as agarose and polyacrylamide. All of these carriers possess more or less hydroxyl-groups on their surface which are responsible for the hydrophilic characteristics and therewith, low non-specific binding.
Prerequisites for the binding of biologically active substances, such as proteins, lectines, enzymes, nucleic acids, low-molecular ligands, cells and other biological materials, are the introduction, in many cases, the introduction of chemically-active groups, which make possible a chemical binding. The chemical modification designated as "activation" depends principally upon the type of functional groups of the matrix and the ligands, but it is also determined from the chemical stability of the mtrix as well as the stability of the biological characteristics of the ligands. The choice of activation technique is moreover, determined by the technological expense of the activation, and the therewith connected cost of the activation technique, the reactivity of the introduced chemical groups, the possibility for the storage of the activated carrier, the toxicity and biocompatibility of modifcation reagents, as well as the stability of the chemical binding between the solid body surface and the ligand.
Accordingly, it is not surprising with the plurality of limiting parameters and influential factors, that in connection with the numerous carrier materials, a good many activation techniques have been described. Representative of these is the activation technique employing glutaraldehyde or other homo- or heter-bifunctional reagents, the CNBr-activation, the activation with hydrazine, bisepoxiranes, divinyl sulfone, epichlorohydrine, benzoquinone, carbonyl imidazolones, triazine, derivatives, tosylchlorides, as well as the periodate-oxidation and diazotization (See: J. M. Egly, E. Boschetti: Practical guide for use in affinity chromatography and related techniques, IBF-LKB, 1983).
Another technique of substantial employment is the activation by means of bromocyanide introduced by Axen et al. (N. Axen, J. Porath, S. Ernback: Nature 214 (1967) 1302). Whether or not this technique is unequivocally associated with a broad employment of carrier-fixed substances in biotechnology and biology, it is still burdened with the disadvantages of a relatively not-stable chemical binding between solid body surfaces and ligand (particularly at pH values less than 5 and greater than 10), which can lead to a not-inconsiderable setting free of the bound ligands (leakage), and therewith strongly limit the employment mainly for therapeutic in vivo techniques in medicine, the high toxicity of bromocyanide, which economically handicaps the technology of activation, and the danger of formation of cationic groups on the carrier. Disadvantages of the hydrazine activation are the technical problems occurring therewith, as well as the low coupling yields with high-molecular ligands.
Other activation techniques, in contrast, are subject to the disadvantages of the high reagent toxicity (divinyl sulfone, triazine derivatives, epichlorohydrine) and/or high cost of the process (divinyl sulfone, tosylchloride, tresylchloride), unfavorable milieu conditions during the coupling, and therewith the danger of an inactivation of biological materials (Bisepoxirane, Triazine Derivative, Epichlorohydrine), a non-stable binding at high pH values (Divinylsulfone, carbonyldiimidazole, diazonium compounds), long activation and binding periods for ligands (epichlorohydrine, disepoxirane), technological problems, which moreover influence the cost of the technique (triazine derivatives, tosylchloride, carbonyldiimidazol diazotization), low coupling yields (periodate oxidation) as well as relatively strong non-specific binding, attributable in a recriprocally active manner to charge transfer (reciprocal action) (benzoquinone, triazine derivatives, diazonium compounds).
It has been known for a long time (R. A. Messing, H. H. Weetall: U.S. Pat. No. 3,510,538; H. H. Weetall, N.Y., Elmira; U.S. Pat. No. 3,652,761; H. H. Weetall, Methods in Enzymol. 44 (1976), 134) that the OH-groups on SiO.sub.2 surfaces, preferably glass, are available as starting points for chemical agents, particularly organosilanes with various functional groups. Moreover, these organosilanes have previously been employed exclusively for inorganic carriers and solid body surfaces.
The aim of the invention is to develop a simple, cost-favorable, non-toxic activation technique for hydroxyl-group-containing, natural and synthetic, polymer solid body surfaces, which lead to stable, activated, solid body surfaces, upon which proteins, nucleic acids, low-molecular ligands, cells, microorganisms and other biological materials can be bound with high stability and yield, as well as biocompatibility.